Methods Digest, Vol 71, Issue 13

Jayanta Tarafdar via methods%40net.bio.net (by jayanta_bckv from yahoo.co.in)
Thu Apr 28 07:57:47 EST 2011


Dear Wo,

The  transfection method of human cells using PEI is amazing. Is this method  
applicable in plant tissue transformation? Besides bombardment, Agrobac. 
transformation, is there any new & easy technology  for plant transformation?
 Thanks.

Jayanta 
-------------------------------------------------------------------------------------------

Dr. Jayanta Tarafdar 
Plant Pathologist & Officer in Charge 
AICRP on Tuber Crops (Kalyani Centre), ICAR
Directorate of Research
BC Krishi Viswavidyalaya
Kalyani 741235
West Bengal 




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Sent: Wed, 27 April, 2011 10:34:24 PM
Subject: Methods Digest, Vol 71, Issue 13

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Today's Topics:

  1. Re: Competitive Assay (WS)
  2. Re: Copper-Glycine buffer(Ph-7.4) (WS)
  3. Re: Transfection of Hi5 cells with Fugene (WS)


----------------------------------------------------------------------

Message: 1
Date: Tue, 26 Apr 2011 13:03:42 -0700 (PDT)
From: WS <novalidaddress from nurfuerspam.de>
Subject: Re: Competitive Assay
To: methods from net.bio.net
Message-ID:
    <067e06b0-ca36-49c3-bb6f-29bbf3bec1dd from r20g2000yqd.googlegroups.com>
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Hi Juan,

if you want to have competition, you have to mix your ligands *before*
you add them to your cells. If you add one of them first, it binds
well to the receptor (a few socend are enough) and it will take
indefinite time until an equilibrium is reached when you add the
competitor. But you may use this setup for measuring turnover. Then
it's called a pulse - chase experiment.

Remember to add more (ligand + competitor) than you have receptors.
You want competition, not complete binding.

PS for a new topic, please start a new thread!

Have fun!

Wo


On Apr 26, 3:53 pm, "Juan Li" <j... from custombiologics.com> wrote:
> Hello everyone,
>
> I would like to run a competitive assay between unlabeled ligand and
> FITC-labeled ligand. How should the 2 compounds be incubated, together in a
> single incubation step or one after another? What's the advantage or
> disadvantage of them?
>
> Any suggestions?
>
> Thanks!
> Juan



------------------------------

Message: 2
Date: Tue, 26 Apr 2011 13:04:54 -0700 (PDT)
From: WS <novalidaddress from nurfuerspam.de>
Subject: Re: Copper-Glycine buffer(Ph-7.4)
To: methods from net.bio.net
Message-ID:
    <4a4c3fed-7d3b-4e89-8297-371ee94fd2ea from n10g2000yqf.googlegroups.com>
Content-Type: text/plain; charset=ISO-8859-1

Yes Jay, you're right. I overlooked the high molarities.

Wo


------------------------------

Message: 3
Date: Tue, 26 Apr 2011 14:26:25 -0700 (PDT)
From: WS <novalidaddress from nurfuerspam.de>
Subject: Re: Transfection of Hi5 cells with Fugene
To: methods from net.bio.net
Message-ID:
    <c21451a0-c4f8-4618-8054-f13a9655cbe5 from 32g2000vbe.googlegroups.com>
Content-Type: text/plain; charset=ISO-8859-1

Dear Pepa,

do you have access to a quicker positive control like GFP? If you
don't have a fluorescence microscope, beta galactosidase or -
glucuronidase and similar are suitable genes, too, as long as you can
get the appropriate X-substrate.  The big advantage against westerns
is that you also will get an idea of the transfection efficiency, as
you will see single transfected cells.

Do you have the possibility to test your transfection reagent with
some other cell line to check if it is ok? Are your cells healthy?any
mycoplasma? really Hi5 cells? (some paranoia might be ok :)

You also may dig the archives of this NG for polyethyleneimine (PEI,
or simply find it here: 
http://www.bio.net/bionet/mm/methods/1999-December/080050.html).
You only need to get the right one, it's crucial (see the protocol)
and it's worth a try for sure I think, as it's horribly cheap: You'll
get a whole bathtub full of final transfection reagent which is
sufficient for a whole flock of sheep (see
http://forums.biotechniques.com/viewtopic.php?f=18&t=13968 for
details, it also works on cows), for a price much less than 1 single
commercial kit). BTW, it's at least similar to JetPEI. Other hopefully
useful links are in this post here: 
http://www.bio.net/bionet/mm/methods/2002-May/093201.html

Have fun!

Wo


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